In contrast to our internal environment, the G-l tract, especially the colon, is continuously exposed to a vast number of commensals and their products (e.g., TLR-ligands). The innate immune response of this organ must therefore be tightly regulated to avoid chronic inflammation and organ dysfunction from the constant exposure to luminal microbiota. Recent data has underlined the physiological tasks of TLR in the colon. We recently identified unique regulatory functions of TLR signaling in colonic the colonic mucosa. We hypothesize that TLR signaling in the gut supports a complex relationship between the host and luminal. microbiota that does not exist in other "sterile" organs. To identify the unique colon-specific TLR-initiated molecular events, we propose three SA. In SA-1 we will identify the mechanisms that regulate TLR-induced pro- and anti-inflammatory pathways in the colon using DSS-sensitive and DSS-resistant TLR-related mutants. Inflammatory, biochemical, microbiological and histological parameters of the colon will be determined under quiescent and inflammatory conditions (i.e., DSS administration). In SA-2 we will identify the pro- and anti-inflammatory effects of lamina propria (LP) and splenic (SP) myeloid dendritic cell (MDC) subsets in the mutants identified in SA-1 using DC ablation and adoptive transfer approaches. The effector molecules produced by TLR-activated SP- and LP-MDC will be further analyzed by a functional genomic approach. In SA-3 we will further explore the protective effects mediated by TLR-activated MDC on colonic epithelium. These include: 1) protection of intestinal epithelial cells (lEC) from cell-death and inflammatory insult, 2) increase of epithelial barrier functions of IEC and 3) enhancing the restitution of injured epithelium (IEC). Dissection via diverse biochemical assays, physiological measurements (electrical resistance and paracellular leak), microbiological analysis (EPEC infection) and signaling events (STAT1 vs. STATS) will be employed. These studies will provide insight into protective mechanisms triggered by TLR-commensals interactions, the resulting unique signaling cascade, and the effector molecules that support colonic homeostasis.